Glycosyltransferases Expression Changes in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 Grown on Different Carbon Sources

Leuconostoc mesenteroides strains are common contributors in fermented foods producing a wide variety of polysaccharides from sucrose through glycosyltransferases (GTFs). These polymers have been proposed as protective barriers against acidity, dehydration, heat, and oxidative stress. Despite its presence in many traditional fermented products and their association with food functional properties, regulation of GTFs expression in Ln. mesenteroides is still poorly understood. The strain Ln. mesenteroides ATCC 8293 contains three glucansucrases genes not found in operons, and three fructansucrases genes arranged in two operons, levLX and levC-scrB, a Glycoside-hydrolase. We described the first differential gene expression analysis of this strain when cultivated in different carbon sources. We observed that while GTFs are expressed in the presence of most sugars, they are down-regulated in xylose. We ruled out the regulatory effect of CcpA over GTFs and did not find regulatory elements with a direct effect on glucansucrases in the condition assayed. Our findings suggest that only operon levLX is repressed in xylose by LexA and that both fructansucrases operons can be regulated by the VicK/VicR system and PerR. It is essential to further explore the effect of environmental conditions in Ln. mesenteroides bacteria to better understand GTFs regulation and polymer function.


Introduction
Leuconostoc mesenteroides bacteria are Gram-positive, catalase-negative, non-motile, non-sporogenous, and facultative anaerobic often found as coccoid shape groups arranged in pairs or chains [1]. They may be found in plant roots, vines, and leaves, from where they are transferred to utensils, animal milk, fruits, and vegetables [2]. Ln. mesenteroides has a heterofermentative metabolism that leads mainly to lactic acid, carbon dioxide, and ethanol, but also to products such as acetate, acetoin, diacetyl, or 2,3-butylene glycol, important components of flavor in fermented products [3].
One of the most interesting properties of Ln. mesenteroides strains is their ability to produce polysaccharides when growing in sucrose-rich environments such as sugar mills. The first Ln. mesenteroides strain was isolated from a gelatinous substance commonly observed over machinery surfaces in sugar mills [4]. In the early 20th century, this substance was identified as dextran [5], a glucose homopolysaccharide (HoP). The discovery of the enzymatic nature of dextran synthesis [6] and the application of dextran as a plasma substitute [7] increased the interest in the search for adequate strain/enzyme sources and the optimization of reaction conditions for dextran synthesis. As a consequence, Ln.

Aerobic Culture Conditions
Leuconostoc medium was composed of: yeast extract 20 g/L, K 2 HPO 4 20 g/L, MgSO 4 0.2 g/L, CaCl 2 0.05 g/L, NaCl 0.01 g/L, MnSO 4 0.01 g/L, and FeSO 4 0.01 g/L. The pH of the culture medium was adjusted to 6.9 by adding HCl. Carbon sources were supplemented from filter-sterilized solutions at 59 mM final concentration in the culture medium. Freeze-dried Ln. mesenteroides ATCC 8293 cells were recovered in LM medium with 59 mM of sucrose at 28 • C and 200 rpm in a Jeio-Tech IST-3075 shaker incubator (Lab Companion, Daejeon, Republic of Korea) overnight and then diluted to 0.3 OD in fresh medium with sucrose until mid-exponential phase to prepare glycerol (40% v/v final) stocks that were stored at −80 • C until usage. To modify the carbon source (CS), 200 µL of glycerol stock was plated in Petri dishes with 59 mM of the new CS and incubated at 28 • C in a 10-140 Incubator (Quincy Lab Inc., Burr Ridge, IL, USA). The resulting colonies were repeatedly streaked in fresh Petri dishes with the new CS. Once the strain was adapted to the new CS, 50 mL of culture medium in 250 mL Erlenmeyer flasks were inoculated with the colonies and incubated overnight at 28 • C and 200 rpm in the shaker incubator with 59 mM of the new CS. Next, the culture was diluted to 0.3 OD in 50 mL of fresh medium and cultured again until the start of the stationary phase. Cells were then centrifuged in Eppendorf tubes for 5 min at 13,000× g and 4 • C (FC5515R Ohaus centrifuge, Gießen, Germany) and washed three times with 50 mM pH 6.0 acetate buffer. Fresh cells were used for RNA extraction. Cells were frozen and stored at −20 • C for further analysis. Stored cells were thawed in ice and homogenized in 50 mM pH 6.0 acetate buffer for protein and activity determination. When sucrose was used as CS, dextranase (ENZIQUIM, Mexico City, Mexico) was added to the culture in the last step to facilitate sample processing. The cultures were carried out in triplicate.

Anaerobic Culture Conditions
The cultures in anaerobic conditions were carried out in serum flasks with LM medium. The oxygen in the headspace and the medium was replaced by bubbling each phase with a gas mixture (80% N 2 , 20% CO 2 ) for at least 30 min and immediately sealed with rubber stoppers and aluminum seals. The closed flasks were sterilized by autoclaving at 116 • C for 26 min. A sucrose solution of 1.75 M was prepared and micro-filtered with a 0.22 µm syringe filter (Millex, Bad Langensalza, Germany) under aseptic conditions; then, CO 2 was bubbled to displace oxygen (1 min/mL in the culture medium and 1 min/mL headspace) and immediately sealed with rubber septa and aluminum cap. The sucrose solution was supplemented to the flasks using a sterile syringe to reach 59 mM concentration in the culture medium. The first sealed flask was inoculated from an aerobic culture using sterile syringes, subsequent cultures were inoculated from the immediate last sealed flask. Four serial cultures in sealed flasks were carried out to minimize O 2 interference. When the culture reached the stationary phase, a fresh anaerobic flask was inoculated and the operation was repeated once a 0.3 OD was reached. After the fourth culture, cells were centrifuged as previously described and frozen at −20 • C for further analysis. All flasks were incubated at 28 • C and 200 rpm. The cultures were carried-out in triplicate.

Biomass and pH Determination
Cell growth was followed by measuring optical density at 600 nm (OD600) using a Beckman DU 640 spectrophotometer (Beckman, Brea, CA, USA), a change of 1 unit of optical density at 600 nm was equivalent to 0.44 g of dry matter. The pH changes were followed by measuring 1 mL of culture with pH Lab Meter Model 827 (MetrOhm, Riverview, FL, USA).

Protein Determination
Protein in cell samples was quantified using the Lowry method [40] with modifications. Initially, the Biuret reaction involves the reduction of copper (Cu 2+ to Cu + ) by proteins in alkaline solutions, followed by the enhancement stage, the reduction of the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). After incubation with Folin-Ciocalteu's phenol reagent, samples were centrifuged for 5-10 s and then absorbance was read at 595 nm using a Beckman DU 640 spectrophotometer (Beckman, Brea, CA, USA). The assay was performed in duplicate for each sample.

GTFs Activity Determination
The total transglycosidase activity was quantified following the initial rate of reducing power release with the 3,5-dinitrosalysilic acid method (DNS) [41]. Total activity refers to enzyme activity when using whole harvested cells, including fructansucrase and glucansucrase activity. Determinations were performed by triplicates for each of the three different flask cultures. One unit of GTFs activity was defined as the amount of enzyme that released 1 µmol of reducing sugars (fructose or glucose) per minute. The specific activity of each sample was determined by calculating the rate of total activity per mg of total protein (U/mg total protein). One-way ANOVA with Tukey's test with α < 0.05 was used to determine the significative difference between specific activities using Microsoft Excel 2019 Software.

Zymograms
GTFs activity were detected by in situ production of polymers on gel after SDS-PAGE as previously described [42]. Briefly, SDS-PAGE gels were prepared at 10% as described by Laemmli [43] and completed in a Mighty Small electrophoresis chamber (Amersham Biosciences, Amersham, UK). Samples were mixed with SDS sample buffer minus β-mercaptoethanol and applied to gel wells. Gels were charged with 200 µg of protein. After electrophoretic separation of the proteins, the lanes containing the molecular mass reference were cut and stained with GelCode (Thermofisher, Hanover Park, IL, USA) following the manufacturer's instructions. For in situ detection of GTFs activity, the SDS the lanes containing the sample protein fractions were washed three times with 50 mM pH 6.5, phosphate buffer containing 1% Tween 80 (Merck, Darmstadt, Germany), for protein renaturation, and once with the Tween-free buffer. The renatured gel was incubated in a buffer solution with 100 g/L of sucrose overnight at 28 • C. Then, the gels were incubated for 30 min in a 75% ethanol solution, followed by incubation for 1 h in 0.7% periodic acid (Sigma-Aldrich, St. Louis, MO, USA) and 5% acetic acid (Mallinckrodt, Staines-Upon-Thames, Surrey, UK) water solution. Next, the gels were washed three times with 0.2% sodium metabisulfite (J.T. Baker S.A. de C.V., Estado de México, Mexico) and 5% acetic acid solutions for 20 min and finally exposed to the Schiff's reagent (Sigma-Aldrich, St. Louis, MO, USA) to stain the polymer produced.

RNA-seq Analysis
RNA-seq analysis was performed with three biological replicates for each carbon source. RNA extraction, quality analysis, and sequencing were performed at UUSMB (Unidad Universitaria de Secuenciación Masiva y Bioinformática de la UNAM) facility. The RNA extraction was performed from three biological replicates with the Quick-RNA miniprep kit (Zymo Research, Irvine, CA, USA) following the manufacturer's protocol from 10 mL of freshly harvested cells. After the integrity and quality analysis were performed (Bioanalyzer 2100, Agilent, Santa Clara, CA, USA) the RNA library was prepared with a Zymo-Seq Ribo Free Total RNA Library kit (Zymo Research) following the manufacturer's protocol. Sequencing was performed in an Illumina Genome Analyzer II (Illumina, San Diego, CA, USA). We obtained two files from each sample.

Motif Search
From the Ln. mesenteroides ATCC 8293 complete genome (GenBank assembly accession: GCA_000014445.1), we extracted the intergenic upstream region of each GTF gene from the start site using the Artemis software (v.18.2.0) [48]. From the extracted upstream regions, we searched the binding site motifs of the regulatory elements CcpA, LexA, and VicR using the FIMO [49] tool from MEME-suite (v.5.4.1) [50], with the default parameters. The CcpA binding site sequence of Lactobacillaceae WTGWAARCGYTTWCAW (from the regulon of CcpA in https://regprecise.lbl.gov/regulon.jsp?regulon_id=34354, consulted on 18 September 2022, where W is A or T and H is A, T, or C) was used as input motif. In the case of the LexA binding site, the regulatory sequences from the regulon of LexA for Ln. mesenteroides subsp. mesenteroides ATCC 8293 (https://regprecise.lbl.gov/regulon.jsp? regulon_id=46374, consulted on 18 September 2022) were used as input motifs. Finally, for VicR, the conserved binding site TGTWAHNNNNNTGTWAH [51], where W is A or T and H is A, T, or C as in gtfC promoter in Strep. mutans U159 was used as the input motif.

RT-qPCR
RNA extraction, cDNA synthesis, and RT-qPCR assays were performed as previously reported [52] with some variations. Five milliliters of cultures in the early stationary phase in each carbon source were centrifuged to harvest fresh cells, and RNA was isolated from the fresh cells using Trizol reagent (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer's instructions. RNA integrity was verified by electrophoresis and the concentration was assessed using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). To remove DNA contamination, the RNA samples were incubated with RNase-free DNase (1 U/µL; Roche, Basel, Switzerland) for 1 h at 37 • C as indicated by the manufacturer. Complementary DNA (cDNA) was synthesized using Thermo Scientific RevertAid Reverse Transcriptase (200 U/µL, Thermo Scientific, Waltham, MA, USA), with 200 ng of DNA-free RNA as a template and following the manufacturer's instructions. RT-qPCR assays were performed using Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific, Waltham, MA, USA), in a real-time PCR system  Table S1. The relative expression values were calculated using the geometric mean of Ct in the ∆∆Ct method [53] using Microsoft Excel 2019 Software. This analysis was performed with three biological replicates for each carbon source and by triplicate. Data available in the Supplementary Materials File S3 and File S4.

Specific Total Activity Decreases in CS Different from Sucrose
We first analyzed the production of GTFs when Ln. mesenteroides ATCC 8293 was cultured in several carbon sources: sucrose, commonly accepted as the inductor [22,34], glucose, and fructose. We expanded the study to alternative carbon sources that the strain can metabolize such as xylose, galactose, mannose, cellobiose, and ascorbic acid, since these compounds are common in plants and therefore, eventual CS in natural raw materials fermentation. As expected, the CSs were consumed with different specificities, as concluded from the different biomass yields (OD 600 ) reached in the stationary phase ( Figure 1). We compared the level of total specific GTF activity produced in each CS, observing a significative decrease when carbon sources other than sucrose were used in the culture medium (Tukey's test with α = 0.05). As already reported for Ln. mesenteroides [21,22,26], sucrose is not the exclusive inductor for GTFs expression, and considerable enzyme activity is detected when the strain is cultured in alternative carbon sources.
incubated with RNase-free DNase (1 U/µL; Roche, Basel, Swi erland) for 1 h at 37 °C as indicated by the manufacturer. Complementary DNA (cDNA) was synthesized using Thermo Scientific RevertAid Reverse Transcriptase (200 U/µL, Thermo Scientific, Waltham, MA, USA), with 200 ng of DNA-free RNA as a template and following the manufacturer's instructions. RT-qPCR assays were performed using Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific, Waltham, MA, USA), in a real-time PCR system (QuantStudio 5; Applied Biosystems, Waltham, MA, USA) following the thermal program: an initial 95 °C for 10 min cycle, 40 cycles of 95 °C for 15 s, and a final 60 °C for 60 s cycle. The dnaX gene was used for normalization. Primers sequences for GTFs and dnaX are summarized in Supplementary Materials Table S1. The relative expression values were calculated using the geometric mean of Ct in the ∆∆Ct method [52] using Microsoft Excel 2019 Software. This analysis was performed with three biological replicates for each carbon source and by triplicate. Data available in the Supplementary Materials File S3 and File S4.

Specific Total Activity Decreases in CS Different from Sucrose
We first analyzed the production of GTFs when Ln. mesenteroides ATCC 8293 was cultured in several carbon sources: sucrose, commonly accepted as the inductor [22,34], glucose, and fructose. We expanded the study to alternative carbon sources that the strain can metabolize such as xylose, galactose, mannose, cellobiose, and ascorbic acid, since these compounds are common in plants and therefore, eventual CS in natural raw materials fermentation. As expected, the CSs were consumed with different specificities, as concluded from the different biomass yields (OD600) reached in the stationary phase (Figure 1). We compared the level of total specific GTF activity produced in each CS, observing a significative decrease when carbon sources other than sucrose were used in the culture medium (Tukey's test with α = 0.05). As already reported for Ln. mesenteroides [21,22,26], sucrose is not the exclusive inductor for GTFs expression, and considerable enzyme activity is detected when the strain is cultured in alternative carbon sources.  We analyzed the produced enzymes in each carbon source through zymograms to explore closely the strain performance. As shown in Figure 2, in sucrose cultures, four bands are obtained in the zymogram, the upper band corresponding to the 308 kDa dextransucrase (defined as DsxD); two activity bands of around 180 kDa corresponding to dextransucrases DsrD, and DsrI, and a fourth activity band of around 130 kDa, associated to levansucrases LevC, LevL, and LevX, which have very similar molecular mass. Surprisingly, when comparing the zymogram profile obtained from cultures with different carbon sources, differences and similarities in the band pattern are observed, in particular, the lack of the DsxD band in cultures with xylose (Xyl) and ascorbic acid (Asc). These compounds, unlike the other carbon sources, share a particular carbohydrate metabolic pathway: the pentose interconversion.
We analyzed the produced enzymes in each carbon source through zymograms to explore closely the strain performance. As shown in Figure 2, in sucrose cultures, four bands are obtained in the zymogram, the upper band corresponding to the 308 kDa dextransucrase (defined as DsxD); two activity bands of around 180 kDa corresponding to dextransucrases DsrD, and DsrI, and a fourth activity band of around 130 kDa, associated to levansucrases LevC, LevL, and LevX, which have very similar molecular mass. Surprisingly, when comparing the zymogram profile obtained from cultures with different carbon sources, differences and similarities in the band pa ern are observed, in particular, the lack of the DsxD band in cultures with xylose (Xyl) and ascorbic acid (Asc). These compounds, unlike the other carbon sources, share a particular carbohydrate metabolic pathway: the pentose interconversion. In the pentose interconversion pathway (Figure 3), D-xylose is isomerized and phosphorylated in successive reactions catalyzed by the xylose-isomerase xylA (LEUM_0130), and the xylulokinase xylB (LEUM_0131) to produce xylulose-5P a common metabolic intermediate that can be then converted to D-ribulose-5P and enter the pentose phosphate pathway [53]. Although the regulatory pathway for GTFs in Leuconostoc strains is poorly understood, a preliminary conclusion regarding the expression of DsxD can be made, considering that its regulation in xylose and ascorbate could be related to the pentose interconversion pathway. In the pentose interconversion pathway (Figure 3), D-xylose is isomerized and phosphorylated in successive reactions catalyzed by the xylose-isomerase xylA (LEUM_0130), and the xylulokinase xylB (LEUM_0131) to produce xylulose-5P a common metabolic intermediate that can be then converted to D-ribulose-5P and enter the pentose phosphate pathway [54]. Although the regulatory pathway for GTFs in Leuconostoc strains is poorly understood, a preliminary conclusion regarding the expression of DsxD can be made, considering that its regulation in xylose and ascorbate could be related to the pentose interconversion pathway.

Differential Expression Analysis (DEA): Using Xylose Produces Major Changes in Gene Expression
To explore in detail GTF induction under common carbon sources, we studied how fructose and xylose affect gene expression compared to sucrose as the control condition. As far as we understand, this is the first DEA performed in a Leuconostoc wild-type strain, regarding the GTFs genes' response to carbon sources. Since any single modification in bacterial culture can affect gene expression, we established a rather large cut-off for the Fold-Change in level expression (FC = 4), to narrow the final number of Differentially Expressed Genes (DEGs) to analyze. Therefore, we considered that if LOG 2 (FC) > 2 or <−2, then the gene is up-regulated or down-regulated, respectively. This cut-off permits us to observe the most dramatic changes in gene expression when using xylose and therefore, to make the first approach to understand the regulation in the strain.
When comparing the number of DEGs obtained in cultures with fructose or xylose as carbon source instead of sucrose, we observed the greatest effect for xylose, with a small number of genes affected when fructose was used as carbon source ( Figure 4). As mentioned earlier, this is most probably the consequence of the xylose metabolism through the pentose interconversion pathway.  . Xylose assimilation and its metabolism in the pentose interconversion. Only xylose uses this metabolic pathway from among the CS tested. Up-regulated genes when using xylose: xylT (corresponding to symporter) and the operon xylAB (corresponding to xylose isomerase XylA, and xylulose kinase XylB). XylR is the regulatory element that effects over xylT, xylA and xylB.

Differential Expression Analysis (DEA): Using Xylose Produces Major Changes in Gene Expression
To explore in detail GTF induction under common carbon sources, we studied how fructose and xylose affect gene expression compared to sucrose as the control condition. As far as we understand, this is the first DEA performed in a Leuconostoc wild-type strain, regarding the GTFs genes' response to carbon sources. Since any single modification in bacterial culture can affect gene expression, we established a rather large cut-off for the Fold-Change in level expression (FC = 4), to narrow the final number of Differentially Expressed Genes (DEGs) to analyze. Therefore, we considered that if LOG2(FC) > 2 or <−2, then the gene is up-regulated or down-regulated, respectively. This cut-off permits us to observe the most dramatic changes in gene expression when using xylose and therefore, to make the first approach to understand the regulation in the strain.
When comparing the number of DEGs obtained in cultures with fructose or xylose as carbon source instead of sucrose, we observed the greatest effect for xylose, with a small number of genes affected when fructose was used as carbon source ( Figure 4). As mentioned earlier, this is most probably the consequence of the xylose metabolism through the pentose interconversion pathway. . Xylose assimilation and its metabolism in the pentose interconversion. Only xylose uses this metabolic pathway from among the CS tested. Up-regulated genes when using xylose: xylT (corresponding to symporter) and the operon xylAB (corresponding to xylose isomerase XylA, and xylulose kinase XylB). XylR is the regulatory element that effects over xylT, xylA and xylB.  Leuconostoc mesenteroides bacteria are selective in terms of the carbohydrate used as a carbon source; hence, there are specific carbohydrate transport systems classified in their soft-core genome [24]. Unfortunately, these systems are still poorly characterized so, for instance, in the Ln. mesenteroides ATCC 8293 annotated genome, 31 genes are identified as "generic transporters belonging to the Major Facilitator Superfamily (MFS)". In unpublished experimental results obtained in our group, we speculated that sucrose, glucose, and fructose could be transported into the cell by the mannose PTS. However, when examining the transporters expression level, we found one up-regulated membrane transporter when using fructose (LEUM_0853) and a different one up-regulated in xylose (LEUM_0128). These findings indicate that fructose and xylose enter the cell through membrane symporters from the MFS.
As expected, genes related to xylose transport and metabolism are highly up-regulated when Ln. mesenteroides ATCC 8293 uses xylose as CS (Figure 3). These genes correspond to the xylose symporter xylT (LEUM_0128, LOG2FC = 4.8), the xylose-isomerase xylA (LEUM_0130, LOG2FC = 5.9), the xylulokinase xylB (LEUM_0131, LOG2FC = 4.3). It is most probable than xylA and xylB genes constitute the xylAB operon, since their expression level is similar in this condition. The transcriptional factor xylR (LEUM_0129, Leuconostoc mesenteroides bacteria are selective in terms of the carbohydrate used as a carbon source; hence, there are specific carbohydrate transport systems classified in their soft-core genome [24]. Unfortunately, these systems are still poorly characterized so, for instance, in the Ln. mesenteroides ATCC 8293 annotated genome, 31 genes are identified as "generic transporters belonging to the Major Facilitator Superfamily (MFS)". In unpublished experimental results obtained in our group, we speculated that sucrose, glucose, and fructose could be transported into the cell by the mannose PTS. However, when examining the transporters expression level, we found one up-regulated membrane transporter when using fructose (LEUM_0853) and a different one up-regulated in xylose (LEUM_0128). These findings indicate that fructose and xylose enter the cell through membrane symporters from the MFS.
As expected, genes related to xylose transport and metabolism are highly up-regulated when Ln. mesenteroides ATCC 8293 uses xylose as CS (Figure 3). These genes correspond to the xylose symporter xylT (LEUM_0128, LOG 2 FC = 4.8), the xylose-isomerase xylA (LEUM_0130, LOG 2 FC = 5.9), the xylulokinase xylB (LEUM_0131, LOG 2 FC = 4.3). It is most probable than xylA and xylB genes constitute the xylAB operon, since their expression level is similar in this condition. The transcriptional factor xylR (LEUM_0129, LOG 2 FC = 1.14) has a repressor effect on genes xylT, xylA, and xylB according to RegPrecise database [55], and, therefore, their expression level is different compared to neighbor genes.
In Table 1 we observe a clear decrease in the expression level of all GTF genes when fructose or xylose are used instead of sucrose as carbon source, supporting the established hypothesis that sucrose is the strongest expression inductor. These results are also consistent with the overall decrease in total activity reported in Figure 1, more evident in xylose than in fructose cultures. Similarly, these results match the qualitative observation from the image analysis of the zymogram in Figure 2, where the intensity of the activity bands corresponding to the sucrose culture sample is rather similar to that of the fructose sample but dimmer in the xylose culture activity bands. Since we used the same amount of total protein for the zymograms, together with the transcriptomic results, we may conclude that dimmer bands correspond to less enzyme quantity and activity, therefore, less expression. A deeper analysis of the Ln. mesenteroides ATCC 8293 genome could provide complementary information regarding operons and regulatory boxes that can be used in future research to expand the regulatory network. We think that additional biochemical and molecular approaches, such as pull-down and DNA affinity chromatography, must be used to prove these findings. In an overall view of GTF expression in cultures containing fructose as carbon source, an interesting observation may be derived based on our original hypothesis. In effect, there is an overall decrease in the expression level of all GTF genes when fructose is used as the carbon source, but with the largest effect in levX, levL, and levC the fructansucrases genes. This may result in a lower content of the overall fructansucrase activity, and eventually, a lower amount of fructans compared to glucans if sucrose is available for polysaccharide synthesis after the fermentation process carried out with fructose as carbon source. Eventually, this may also become a strategy to direct cultures of this strain towards the synthesis of diverse fructan-free glucans.

Exploring Possible Regulation Pathways of Leuconostoc mesenteroides ATCC 8293 GTFs
Although sucrose has been generally recognized as the inductor in GTFs expression in Leuconostoc strains, the mechanism behind GTFs regulation has yet to be demonstrated [22]. Very few works deal in detail with the eventual physiological mechanism; instead, most reports related to regulation limit their scope to report the differences in total GTF activity in response to environmental changes, such as carbon source [21], temperature [56,57], or oxygen availability [58,59]. D-xylose is a major component of cell walls in plants, and therefore, a carbon source for Leuconostoc bacteria since their ecological niche is vegetal matter and roots [3]. Based on the large differences in gene expression between cells grown in D-xylose compared to sucrose cultures (xylose/sucrose), we carried out a detailed analysis of these differences as inferred from the known regulatory pathways in Firmicutes species in search of genomic clues of GTFs regulation.
One of the most down-regulated genes found when using D-xylose is pgi encoding a glucose-6-phosphate isomerase, involved in exopolysaccharide (EPS) production in Lactic Acid Bacteria (LAB) species. This is the case for instance of Lactococcus lactis and Strep. thermophilus where Pgi produces hexose-phosphate as the substrate to synthesize sugar nucleotides, the EPS precursors [60]. However, these are EPS involving Leloir GTFs in their synthesis, requiring high-energy activated sugars as substrate for transglycosylation [17]. Consequently, their regulation can be related to the energy metabolism and pgi expression. Considering the zymogram results, it is possible to relate pgi with regulation, at least for the dsxD gene. Since pgi is regulated by the transcription factor CcpA (Carbon catabolite control protein A), we first explored this regulation element.

Genomic Context of GTFs Genes
The total length of the sequenced genome of the Ln. mesenteroides ATCC 8293 strain is 2.07 Mb. It consists of 2047 genes including the GTFs genes. When analyzing the localization of the GTFs genes in the strain genome, we observed that the glucansucrases genes (dsxD, dsrD, and dsrI) are scattered through the genome, and as concluded through the Operon Mapper software analysis [47] these genes are neither located in operons ( Figure 5A). On the other hand, the fructansucrase genes are located in the negative strand, within the same region in the genome ( Figure 5B) and arranged in two operons: levLX and levC-scrB. The scrB gene also encodes a sucrose-6-phosphate hydrolase. Given that fructansucrases are arranged in operons, we used the upstream intergenic region of operon levLX and levC-scrB for the region analysis. 5A). On the other hand, the fructansucrase genes are located in the negative strand, within the same region in the genome ( Figure 5B) and arranged in two operons: levLX and levC-scrB. The scrB gene also encodes a sucrose-6-phosphate hydrolase. Given that fructansucrases are arranged in operons, we used the upstream intergenic region of operon levLX and levC-scrB for the region analysis.

The Transcription Factor (TF) CcpA
The protein CcpA plays a central role in the regulation of the Phosphoenolpyruvatedependent Transferase System (PTS), the main pathway for carbohydrate uptake in bacteria [60] and controls carbohydrate metabolism in several species. CcpA belongs to the LacI-GalR family of bacterial TFs which represses gene expression by allosteric hindrance [61]. CcpA forms a complex with phosphorylated HPr to be able to bind the gene promotor region preventing transcription. The DNA sequence where the complex CcpA-Hpr-P binds to the promotor is known as the catabolite response element (cre), and its canonical sequence has been identified for Lactobacillaceae (WTGWAARCGYTTWCAW) through experimental and comparative genomics [54,60]. Previously, in unpublished research of our

The Transcription Factor (TF) CcpA
The protein CcpA plays a central role in the regulation of the Phosphoenolpyruvatedependent Transferase System (PTS), the main pathway for carbohydrate uptake in bacteria [61] and controls carbohydrate metabolism in several species. CcpA belongs to the LacI-GalR family of bacterial TFs which represses gene expression by allosteric hindrance [62].
CcpA forms a complex with phosphorylated HPr to be able to bind the gene promotor region preventing transcription. The DNA sequence where the complex CcpA-Hpr-P binds to the promotor is known as the catabolite response element (cre), and its canonical sequence has been identified for Lactobacillaceae (WTGWAARCGYTTWCAW) through experimental and comparative genomics [55,61]. Previously, in unpublished research of our group, we were not able to locate the cre binding site within the promoter region of GTFs from Ln. mesenteroides ATCC 8293, discarding the participation of CcpA in GTFs regulation. To update our findings, we analyzed the upstream intergenic region of each gene with the FIMO tool from MEME-suite [49,50] in a more detailed search for the canonical cre site. Again, the cre binding site was not located. Hence, we may conclude that in the strain Ln. mesenteroides ATCC 8293, the GTFs genes are not regulated by CcpA. Furthermore, when analyzing the transcription levels of the PTS components in our differential expression analysis on xylose/sucrose, we found that the genes: ccpA (LEUM_0544, LOG 2 FC = −1.2), ptsH (corresponding to HPr protein; LEUM_1780, LOG 2 FC = −1.45), and scrA (corresponding to the phosphate-kinase; LEUM_0508, LOG 2 FC = −3.7) are down-regulated, suggesting a minor quantity of CcpA protein and thus, limiting their activity as repressors under these culture conditions.

The Transcription Factor PerR
Recent work suggests that GTFs expression in Ln. mesenteroides BD3749 strain responds to the environmental oxygen level; since expression of the GTF, identified as Gsy in this strain, increases as a consequence of Reactive Oxygen Species (ROS) accumulation, the synthesized polymer may then act as cell protector absorbing ROS [59]. These results allow us to speculate that a key to GTFs regulation could be ROS since Leuconostoc strains lack catalase, requiring the H 2 O 2 sensor PerR to deal with oxidation stress. PerR is a dimeric protein that in its metal-bound form binds DNA, preventing the expression of the regulated genes; when H 2 O 2 levels rise, a series of reactions are triggered, which induces a conformational change in the protein, preventing binding to DNA [63].
In our DEA, perR (LEUM_0492) is down-regulated in xylose/sucrose (LOG 2 FC = −2.3), limiting its activity as a repressor, as already described for CcpA. Furthermore, we analyzed growth and total GTF activity in cultures obtained in anaerobic conditions compared to aerobic cultures. Contrary to results reported for Ln. mesenteroides BD3749, Ln. mesenteroides ATCC 8293 does not show differences in total GTF total specific activity or growth in cultures obtained either in aerobic or anaerobic conditions (Table 2). Furthermore, in an additional report dealing with Ln. mesenteroides YL48 the authors claim that, in terms of regulation response to ROS, GTF up-regulation occurs in oxygen-depleted cultures with high CO 2 levels [58]. The ensemble of results regarding ROS response suggests that the regulation of GTFs can be particular for each strain.

The Transcription Factor LexA
Recent work suggests that GTFs in Weissella cibaria C2-32 may be regulated by TF LexA, a protein involved in the SOS response system [57]. In Escherichia coli growing in normal conditions, dimeric LexA binds to the DNA strand and represses DNA repair genes; meanwhile, in severe culture conditions, such as low pH-, LexA forms aggregates with a lower capacity to repress transcription [64]. In our transcriptomic data, lexA in xylose/sucrose was up-regulated (LEUM_1204, LOG 2 FC = 2.4), while its expression remains similar in fructose/sucrose (LEUM_1204, LOG 2 FC = 0.1). This led us to search for the LexA binding site sequence in the GTFs upstream regions using the FIMO tool [49], finding one match in the upstream sequence of the levLX operon. These preliminary findings suggest that levLX operon is repressed by LexA when Ln. mesenteroides grows on xylose as a carbon source. Interestingly, the final pH, reached in the stationary phase is lower when using sucrose (pH = 4.53), a bit higher in fructose (pH = 5.26), and even higher in xylose (pH = 6.13). Nevertheless, this implies that the low pH in the final stage of the culture may become a severe condition for the strain ATCC 8293, affecting LexA's ability to bind to DNA to allow the transcription of the levLX operon.

VicK/VicR Two-Component System
There is an enormous interest in understanding the regulation of GTFs in Streptococccus mutans since these enzymes allow the bacteria the synthesis of the biofilm that enables cell adhesion to teeth, the initial step that leads to dental plaque formation and caries in humans [65]. This biofilm is mainly formed by polysaccharides synthesized by GTFs from sucrose, so their activity is considered a virulence factor [19,20]. GTFs enzymes from Strep. mutans and Ln. mesenteroides are orthologous proteins with high sequence identity (48%); as both species belong to the Lactobacillales order, they may share regulatory pathways. In Strep. mutans, the two-component system vicRK (formerly covRK) regulates the transcription of gtfBCD operon (where GTFs genes are located) in response to temperature stress-VicK auto-phosphorylates at high temperatures, transferring a phosphate group to VicR, that then binds to the promoter region of regulated genes to repress its expression [66]. Searching in the DEA, we found that the level of expression of the vicRK system remains constant in our experiments and comparisons (xylose/sucrose and fructose/sucrose). Consequently, we concluded that vicRK is not responsible for the observed changes in GTFs expression in cultures grown with different carbon sources. However, when we searched for the VicR binding site TGTWAHNNNNNTGTWAH [51] (where W is A or T and H is A, T, or C) in the GTFs intergenic upstream sequences using FIMO, we obtained regions for possible binding sites in the fructansucrases operons (Table 3). These findings suggest that fructansucrases genes regulation may be affected by temperature stress, another adverse condition that must be explored to understand their role in GTFs regulation.

Transcriptomics Validation by Retro-Transcriptase qPCR
We validated our transcriptomics results with a qPCR analysis of the GTFs with the dnaX gene as housekeeping (data in Supplementary Materials File S3 and File S4). We used fructose as the control condition since we observed no significant difference in dnaX expression between fructose and sucrose, and also because no EPS is produced in cultures with fructose as CS. In general, the RT-qPCR showed the same expression pattern found in the transcriptomics analysis: levC, levX, levL, dsrI, and dsxD are down-regulated, while there is no significant difference (t-test with α = 0.05) in dsrD expression between the conditions essayed ( Table 4). As a result of the RT-qPCR experiments, we observed a consistent down-regulation in GTFs expression when modifying the CS, as already observed through transcriptomics, validating the former.

Conclusions
The scarce information concerning GTF regulation makes it difficult to assemble a possible general regulatory network. We contribute to the task by analyzing known regulatory pathways from GTFs Firmicutes producers in the context of our DEA experiments. A deeper analysis of the Ln. mesenteroides ATCC 8293 genome could provide additional information regarding operons and regulatory boxes that may be used in new searches to expand the knowledge regarding the regulatory network. The availability of nutraceutical bioactive polysaccharides during fermentation through regulation of GTFs expression may enhance the symbiotic properties of characteristics of fermented foods. This in turn may be achieved by controlling certain aspects of traditional food fermentations such as temperature, pH, carbon availability, or oxygen content. Additional biochemical and molecular approaches, such as pull-down and DNA affinity chromatography, together with adverse environmental conditions can be the key to understanding the regulation of GTFs and must be used to validate our findings. More regulation information requires the expression quantification (qPCR) and production of each GTF (quantitative proteomics) in cultures obtained from different carbon sources, exploring also the effect of additional culture conditions such as temperature, pH, oxidative stress (oxygen, H 2 O 2 , and CO 2 effect), in this and additional strains bearing diverse GTFs genes.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods12091893/s1, Table S1: Primers used for qPCR. File S1: transcript abundances and LOG 2 FC for xylose/sucrose conditions. File S2: transcript abundances and LOG 2 FC for fructose/sucrose conditions. File S3: Ct raw data from the qPCR experiment. File S4: ∆∆Ct calculations for the qPCR experiment.